CN108159022B - Adriamycin and ABT-263 double-drug nano-carrier and preparation method thereof - Google Patents

Adriamycin and ABT-263 double-drug nano-carrier and preparation method thereof Download PDF

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CN108159022B
CN108159022B CN201711436044.9A CN201711436044A CN108159022B CN 108159022 B CN108159022 B CN 108159022B CN 201711436044 A CN201711436044 A CN 201711436044A CN 108159022 B CN108159022 B CN 108159022B
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drug
adriamycin
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stirring
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CN108159022A (en
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李雪萍
李科
徐仓宝
周韵
杨颖�
周连锁
岳波
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Xian Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5084—Mixtures of one or more drugs in different galenical forms, at least one of which being granules, microcapsules or (coated) microparticles according to A61K9/16 or A61K9/50, e.g. for obtaining a specific release pattern or for combining different drugs

Abstract

The invention discloses an adriamycin and ABT-263 double-drug nano-carrier which comprises the following effective components in percentage by mass: 52-60% of medical gelatin, 1.5-3% of ABT-263, 35-42% of hyaluronic acid and 1.5-3% of adriamycin, wherein the sum of the mass percentages of the components is 100%.

Description

Adriamycin and ABT-263 double-drug nano-carrier and preparation method thereof
Technical Field
The invention belongs to the technical field of nano-medicines, and particularly relates to an adriamycin and ABT-263 double-medicine nano-carrier, and a preparation method of the adriamycin and ABT-263 double-medicine nano-carrier.
Background
Chemotherapy achieves therapeutic effect by using drugs to kill tumor cells or inhibit their proliferation, infiltration and metastasis. It has therapeutic effect on primary focus and metastatic focus, which is the only widely used global tumor therapeutic means in clinic today. However, the chemotherapeutic drug has poor selectivity and strong toxicity, and can kill tumor cells and also damage normal tissue cells after entering the body. In addition, drug resistance is another problem that cannot be avoided in chemotherapy, which causes a vicious circle with decreased efficacy, increased dose, and stronger toxic side effects. So that tumor chemotherapy is commonly applied clinically at present by multiple drugs at the same time. Because of the synergistic effect of different medicines, the traditional Chinese medicine composition can show obvious advantages in the aspects of treatment effect, toxic and side effects, dosage control and reduction of drug resistance. However, in the combined chemotherapy, the most critical problem is how to overcome the problem that the synergistic effect of the drugs is difficult to play due to the difference of physicochemical properties and metabolism. Our previous studies found that doxorubicin and the Bcl-2 protein family inhibitor, ABT-263, had significant synergy at the cellular level, but the two drugs were different in hydrophilicity and hydrophobicity, resulting in no significant synergy being exhibited in vivo experiments.
The nano-drug carrier has great advantages in this respect, two or more drugs are integrated on the same carrier by the technology, so that the drugs have no metabolic power difference, can be synchronously delivered into tumor tissues and cells, and can maximally exert the synergistic effect by adjusting the drug in the carrier to the optimal synergistic dose ratio. In order to achieve the series of functions, firstly, the nano-drug carrier needs to be reasonably designed, then, the functionalized wrapping material is prepared according to the requirements, and finally, the complete composite nano-drug carrier is constructed and obtained. The process needs to optimize and reform key nodes by combining actual experience with a new construction process to continuously explore and test.
Disclosure of Invention
The invention aims to provide a double-drug nano-carrier of adriamycin and ABT-263, which can enable two drugs with different physicochemical properties to be accumulated at a tumor part at the same time, thereby playing a role in synergistic treatment.
The invention also aims to provide a preparation method of the adriamycin and ABT-263 double-drug nano-carrier.
The first technical scheme adopted by the invention is that the adriamycin and ABT-263 double-drug nano-carrier consists of the following effective components in percentage by mass: 52 to 60 percent of medical gelatin, 1.5 to 3 percent of ABT-263, 35 to 42 percent of hyaluronic acid and 1.5 to 3 percent of adriamycin, wherein the sum of the mass percentages of the components is 100 percent.
The first technical aspect of the present invention is also characterized in that,
the molecular weight of the hyaluronic acid is 6000-8000 daltons.
The second technical scheme adopted by the invention is that the preparation method of the adriamycin and ABT-263 double-drug nano-carrier is characterized by comprising the following steps:
step 1, weighing 52-60% of medical gelatin, 78-3% of ABT-2631.5, 35-42% of hyaluronic acid and 1.5-3% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding the weighed medical gelatin into 10ml of water, soaking for a period of time, heating and stirring, and cooling to 30 ℃;
step 3, dissolving ABT-263 and polysorbate-80 in ethanol, dropwise adding the solution into the gelatin solution prepared in the step 2, stirring until the solution is turbid, adding a glutaraldehyde solution, increasing the stirring speed, reacting for a period of time in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion, removing an ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion through dialysis, and finally fixing the volume to 18-22ml with water;
step 4, slowly dropping the adriamycin aqueous solution into the hyaluronic acid aqueous solution, stirring for a period of time, adding a glutaraldehyde solution, and keeping out of the sun for a period of time to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to pH 4 with glacial acetic acid, then adding EDC and NHS, stirring uniformly, standing for a period of time, dialyzing the nano drug-loaded nanoparticle dispersion liquid to remove impurities and a solvent, then dropwise adding the ABT-263 gelatin drug-loaded nanoparticle dispersion liquid obtained in the step 3 in a stirring state, stirring, and reacting at room temperature;
and 6, filtering the nano drug-loaded particle dispersion liquid finally obtained in the step 5 to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder.
The second technical aspect of the present invention is also characterized in that,
step 2, using 300-500 mg of gelatin in traditional Chinese medicine, controlling the soaking water temperature at 25 ℃, soaking for 1 hour, heating to 50-55 ℃, and stirring for 20 min.
In the step 3, 10-15 mg of ABT-263, 100-200 mul of polysorbate-80, 12-20ml of ethanol, 25% of glutaraldehyde solution, 200-400 mul of volume, 2-4 ml/min of ABT-263 ethanol solution dropwise adding speed, 200-400 rpm of stirring speed, 800-1000 rpm of stirring speed, 2-4 hours of light-resistant reaction time, and the dialysis is to pack the nano drug-loaded particle dispersion into a dialysis bag with the molecular weight cutoff of 3.5kDa, use pure water as external liquid, 12-24 hours of dialysis time and replace the external liquid for 3-5 times.
In the step 4, the concentration of the adriamycin aqueous solution is 10mg/ml, the addition amount is 1-1.5 ml, the concentration of the hyaluronic acid aqueous solution is 2-3 mg/ml, the volume is 100ml, the dropping speed of the adriamycin aqueous solution is 5-10 ml/min, the stirring speed is 800-1500 rpm, the stirring time is 20min, the concentration of the glutaraldehyde solution is 25%, the addition amount is 200-400 mu l, and the duration of keeping out of the sun is 2-4 hours.
In the step 5, the amount of EDC is 40-60 mg, the amount of NHS is 4-6 mg, the standing time is 4-6 hours, the dialysis is to pack the nano drug-loaded particle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, pure water is used as an external liquid, the dialysis time is 12-24 hours, the external liquid is replaced for 3-5 times, the acceleration of the ABT-263 gelatin drug-loaded nano particle dispersion liquid drop is 2-4 ml/min, the stirring speed is 200-400 rpm, and the room-temperature reaction time is 4-8 hours.
And 6, pre-freezing at the ultralow temperature of-70 to-80 ℃ for 12 to 24 hours, and drying by using a freeze dryer under the pressure of less than 0.1 pascal.
The adriamycin and ABT-263 double-drug nano carrier has the advantages of particle size smaller than 300 nanometers, high packaging efficiency, good stability, stable release and the like, can effectively ensure long-acting circulation of drugs in vivo, can obviously reduce the dosage, can be used for combined treatment of tumors of the double drugs, is simple and practical in established process system, can carry out precise quality control in each step, has very high packaging rate and drug-loading rate, and can be stably stored for more than one year after being freeze-dried.
Drawings
FIG. 1 is a schematic flow chart of a preparation method of a doxorubicin and ABT-263 double-drug nano-carrier of the invention;
FIG. 2 is a transmission electron microscope photograph of the nano-preparation prepared in example 1 after redissolving in the method for preparing the adriamycin and ABT-263 double-drug nano-carrier of the invention;
FIG. 3 is a diagram showing the analysis result of the particle size of the nano-preparation prepared in example 1 after redissolving in the method for preparing a dual-drug nano-carrier of adriamycin and ABT-263 according to the present invention;
FIG. 4 is a graph showing the results of particle size stability tests of the nano-formulation solution prepared in example 1 in the method for preparing a dual-drug nano-carrier of adriamycin and ABT-263 according to the present invention;
FIG. 5 shows the results of in vitro anti-tumor experiments of the nano-preparation prepared in example 1 in the method for preparing a dual-drug nano-carrier of adriamycin and ABT-263 according to the present invention;
FIG. 6 shows the in vivo anti-tumor experimental results of the nano-preparation prepared in example 1 in the preparation method of the doxorubicin and ABT-263 double-drug nano-carrier of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The invention relates to an adriamycin and ABT-263 double-drug nano-carrier which comprises the following effective components in percentage by mass: 52-60% of medical gelatin, 1.5-3% of ABT-263, 35-42% of hyaluronic acid and 1.5-3% of adriamycin, wherein the sum of the mass percentages of the components is 100%, and the molecular weight of the hyaluronic acid is 6000-8000 Dalton.
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is shown in figure 1 and specifically comprises the following steps:
step 1, weighing raw materials: weighing 52-60% of medical gelatin, 1.5-3% of ABT-263, 35-42% of hyaluronic acid and 1.5-3% of adriamycin according to mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 300-500 mg of medical gelatin into 10ml of water, soaking for 1 hour at 25 ℃, heating to 50-55 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 10-15 mg of ABT-263 and 100-200 mul of polysorbate-80 into 12-20ml of ethanol, dropwise adding the solution into the gelatin solution prepared in the stirring step 2, wherein the dropwise adding speed of the ABT-263 ethanol solution is 2-4 ml/min, the stirring speed is 200-400 rpm, adding 200-400 mul of 25% glutaraldehyde solution after the solution is turbid, raising the stirring speed to 800-1000 rpm, reacting for 2-4 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion liquid, removing the ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion liquid through dialysis, filling the nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 3.5kDa, taking pure water as an external liquid, controlling the dialysis time to 12-24 hours, replacing the external liquid for 3-5 times during the dialysis, and finally adding water to 18-22ml of constant volume;
step 4, slowly dropping 1-1.5 ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2-3 mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 5-10 ml/min, the stirring speed is 800-1500 rpm, stirring for 20min, adding 200-400 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 2-4 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 40-60 mg of EDC and 4-6 mg of NHS, stirring uniformly, standing for 4-6 hours, then filling the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, dialyzing for 12-24 hours, replacing the external liquid for 3-5 times to remove impurities and solvents, then dropwise adding the ABT-263 drug-loaded nanoparticle dispersion liquid obtained in the step 3 in a stirring state, wherein the dropwise adding speed is 2-4 ml/min, the stirring speed is 200-400 rpm, and reacting for 4-8 hours at room temperature;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, prefreezing, drying and removing water to obtain the final nano preparation dry powder, wherein the prefreezing is carried out at the ultralow temperature of-70 to-80 ℃, the prefreezing time is 12-24 hours, and the drying is carried out by using a freeze dryer under the pressure of less than 0.1 pascal.
Example 1
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is shown in figure 1 and specifically comprises the following steps:
step 1, weighing raw materials: weighing 55% of medical gelatin, 2% of ABT-263, 41% of hyaluronic acid and 2% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 300mg of medical gelatin into 10ml of water, soaking for 1 hour at 25 ℃, heating to 50 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 11mg of ABT-263 and 110 mu l of polysorbate-80 into 12ml of ethanol, dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 2 ml/min and a stirring speed of 200rpm, adding 200 mu l of 25% glutaraldehyde solution after the solution is turbid, raising the stirring speed to 800rpm, reacting for 2 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion, removing an ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion through dialysis, filling the nano drug-loaded particle dispersion into a dialysis bag with the molecular weight cutoff of 3.5kDa, using pure water as an external solution, changing the external solution for 3 times during dialysis, and finally using water to fix the volume to 18 ml;
step 4, slowly dropping 1.1ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2.2mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 5 ml/min, the stirring speed is 800rpm, stirring for 20min, then adding 200 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 2 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 40mg of EDC and 4mg of NHS, stirring uniformly, standing for 4 hours, then filling the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 12 hours, the external liquid is replaced for 3 times to remove impurities and solvents, and then, under the stirring state, dropwise adding the ABT-263 drug-loaded nanoparticle dispersion liquid obtained in the step 3 at a speed of 2 ml/min, a stirring speed of 200rpm, and reacting at room temperature for 4 hours;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-70 ℃, the pre-freezing time is 12 hours, the drying is carried out by using a freeze dryer, and the pressure is controlled to be lower than 0.1 pascal.
Part of the nano-preparation dry powder obtained in the example was dissolved in sterilized pure water, and the particle size was measured by a malvern particle sizer to analyze the properties, and the nano-preparation obtained in the example had an average particle size of 273 ± 16.3nm and a coating rate: ABT-263 is 74.3 +/-5.1%, and DOX is 71.1 +/-3.8%.
Example 2
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is implemented according to the following steps:
step 1, weighing raw materials: weighing 60% of medical gelatin, 1.5% of ABT-263, 37% of hyaluronic acid and 1.5% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 400mg of medical gelatin weighed into 10ml of water, soaking for 1 hour at 25 ℃, heating to 55 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 10mg of ABT-263 and 100 mu l of polysorbate-80 into 12ml of ethanol, dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 4ml/min and a stirring speed of 400rpm, adding 400 mu l of 25% glutaraldehyde solution after the solution is turbid, raising the stirring speed to 1000rpm, reacting for 4 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion, removing the ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion through dialysis, filling the nano drug-loaded particle dispersion into a dialysis bag with the molecular weight cutoff of 3.5kDa, taking pure water as an external solution, carrying out dialysis for 24 hours, replacing the external solution for 5 times, and finally adding water to fix the volume to 19 ml;
step 4, slowly dropping 1ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2.5mg/ml, adding the adriamycin aqueous solution into the solution at the dropping speed of 10 ml/min and the stirring speed of 1000rpm, stirring the solution for 20 minutes, adding 400 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 4 hours to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 60mg of EDC and 6mg of NHS, stirring uniformly, standing for 6 hours, then filling the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 24 hours, the external liquid is replaced for 5 times to remove impurities and solvents, and then, under the stirring state, dropwise adding the ABT-263 drug-loaded nanoparticle dispersion liquid obtained in the step 3 at a speed of 4ml/min, a stirring speed of 400rpm, and reacting at room temperature for 8 hours;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-80 ℃, the pre-freezing time is 24 hours, the drying is carried out by using a freeze dryer, and the pressure is controlled to be lower than 0.1 pascal.
Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a malvern particle sizer to analyze the properties, and the nano-preparation obtained in this example had an average particle size of 266 ± 12.7nm, a coating rate: ABT-263 is 71.4 +/-5.3%, and DOX is 74.3 +/-4.1%.
Example 3
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is implemented according to the following steps:
step 1, weighing raw materials: weighing 60% of medical gelatin, 2.5% of ABT-263, 35% of hyaluronic acid and 2.5% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 360mg of medical gelatin into 10ml of water, soaking for 1 hour at 25 ℃, heating to 52 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 15mg of ABT-263 and 150 mu l of polysorbate-80 into 20ml of ethanol, dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 3 ml/min and a stirring speed of 400rpm, adding 300 mu l of 25% glutaraldehyde solution after the solution is turbid, raising the stirring speed to 900rpm, reacting for 3 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion, removing the ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion through dialysis, filling the nanoparticle dispersion into a dialysis bag with a molecular weight cutoff of 3.5kDa, using pure water as an external solution, dialyzing for 18 hours, replacing the external solution for 4 times, and finally adding water to fix the volume to 20 ml;
step 4, slowly dropping 1.5ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2.1mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 8 ml/min, the stirring speed is 1200rpm, stirring for 20min, then adding 300 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 3 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 50mg of EDC and 5mg of NHS, stirring uniformly, standing for 5 hours, then filling the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 20 hours, the external liquid is replaced for 4 times to remove impurities and solvents, and then, under the stirring state, dropwise adding the ABT-263 drug-loaded nanoparticle dispersion liquid obtained in the step 3 at the speed of 3 ml/min, the stirring speed of 300rpm, and reacting at room temperature for 6 hours;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-75 ℃, the pre-freezing time is 20 hours, the drying is carried out by using a freeze dryer, and the pressure is controlled to be lower than 0.1 pascal.
Part of the nano-preparation dry powder obtained in the example was dissolved in sterilized pure water, and the particle size was measured by a malvern particle sizer to analyze the properties, and the nano-preparation obtained in the example had an average particle size of 286 ± 13.1nm and a coating rate: ABT-263 is 70.9 +/-5.3%, and DOX is 73.7 +/-7.7%.
Example 4
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is implemented according to the following steps:
step 1, weighing raw materials: weighing 52% of medical gelatin, 3% of ABT-263, 42% of hyaluronic acid and 3% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 260mg of medical gelatin into 10ml of water, soaking for 1 hour at 25 ℃, heating to 54 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 15mg of ABT-263 and 120 mu l of polysorbate-80 into 15ml of ethanol, dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 2 ml/min and a stirring speed of 200rpm, adding 200 mu l of 25% glutaraldehyde solution after the solution is turbid, raising the stirring speed to 800rpm, reacting for 2 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion, removing an ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion through dialysis, filling the nano drug-loaded particle dispersion into a dialysis bag with the molecular weight cutoff of 3.5kDa, using pure water as an external solution, carrying out dialysis for 18 hours, replacing the external solution for 4 times, and finally adding water to fix the volume to 21 ml;
step 4, slowly dropping 1.5ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2.1mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 7 ml/min, the stirring speed is 900rpm, stirring for 20min, then adding 250 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 3 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 45mg of EDC and 4.5mg of NHS, stirring uniformly, standing for 5 hours, then loading the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 18 hours, the external liquid is replaced for 4 times to remove impurities and solvents, then dropwise adding the ABT-263 drug-loaded gelatin nanoparticle dispersion liquid obtained in the step 3 under the stirring state, wherein the dropwise adding speed is 4ml/min, the stirring speed is 300rpm, and reacting at room temperature for 5 hours;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-70 ℃, the pre-freezing time is 18 hours, the drying is carried out by using a freeze dryer, and the pressure is controlled to be lower than 0.1 pascal.
Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a malvern particle sizer to analyze the properties, and the nano-preparation obtained in this example had an average particle size of 291 ± 14.5nm, an inclusion ratio: ABT-263 is 70.2 +/-6.8%, and DOX is 71.7 +/-8.1%.
Example 5
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is implemented according to the following steps:
step 1, weighing raw materials: weighing 58% of medical gelatin, 1.2% of ABT-263, 38% of hyaluronic acid and 1.2% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 348mg of medical gelatin into 10ml of water, soaking for 1 hour at 25 ℃, heating to 54 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 12mg of ABT-263 and 150 mu l of polysorbate-80 into 18ml of ethanol, dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 2 ml/min and a stirring speed of 200rpm, adding 200 mu l of 25% glutaraldehyde solution after the solution is turbid, raising the stirring speed to 1000rpm, reacting for 4 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion, removing the ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion through dialysis, filling the nanoparticle dispersion into a dialysis bag with the molecular weight cutoff of 3.5kDa, using pure water as an external solution, changing the external solution for 4 times during dialysis, and finally using water to fix the volume to 22 ml;
step 4, slowly dropping 1.2ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2.3mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 7 ml/min, the stirring speed is 1300rpm, stirring for 20min, then adding 350 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 2 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 55mg of EDC and 5mg of NHS, stirring uniformly, standing for 4 hours, then filling the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 20 hours, the external liquid is replaced for 4 times to remove impurities and solvents, and then, under the stirring state, dropwise adding the ABT-263 drug-loaded nanoparticle dispersion liquid obtained in the step 3 at a speed of 3 ml/min, a stirring speed of 400rpm, and reacting at room temperature for 7 hours;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-70 ℃, the pre-freezing time is 16 hours, and the drying is carried out by using a freeze dryer under the control of the pressure lower than 0.1 pascal.
Part of the nano-preparation dry powder obtained in this example was dissolved in sterilized pure water, and the particle size was measured by a malvern particle sizer to analyze the properties, and the nano-preparation obtained in this example had an average particle size of 276 ± 11.3nm, a coating rate: ABT-263 is 78.3 +/-5.0%, and DOX is 75.3 +/-6.1%.
Example 6
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is implemented according to the following steps:
step 1, weighing raw materials: weighing 55% of medical gelatin, 13% of ABT-263, 39.5% of hyaluronic acid and 2.5% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 320mg of medical gelatin weighed into 10ml of water, soaking for 1 hour at 25 ℃, heating to 54 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 12mg of ABT-263 and 180 mu l of polysorbate-80 into 16ml of ethanol, dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 4ml/min and a stirring speed of 350rpm, 300 mu l of 25% glutaraldehyde solution is added after the solution is turbid, the stirring speed is increased to 800rpm, and the solution is reacted for 2 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion liquid, removing the ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion liquid through dialysis, filling the nano drug-loaded particle dispersion liquid into a dialysis bag with the molecular weight cutoff of 3.5kDa, taking pure water as an external liquid, carrying out dialysis for 18 hours, replacing the external liquid for 5 times, and finally adding water to fix the volume to 21 ml;
step 4, slowly dropping 1ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2.4mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 8 ml/min, the stirring speed is 1400rpm, stirring for 20min, adding 300 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 4 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 50mg of EDC and 4.5mg of NHS, stirring uniformly, standing for 6 hours, then loading the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 18 hours, the external liquid is replaced for 5 times to remove impurities and solvents, and then dropwise adding the ABT-263 drug-loaded gelatin nanoparticle dispersion liquid obtained in the step 3 under the stirring state, wherein the dropwise adding speed is 3 ml/min, the stirring speed is 350rpm, and reacting at room temperature for 6 hours;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-75 ℃, the pre-freezing time is 12-24 hours, and the drying is carried out by using a freeze dryer under the control of the pressure lower than 0.1 pascal.
Part of the nano-preparation dry powder obtained in the example was dissolved in sterilized pure water, and the particle size was measured by a malvern particle sizer to analyze the properties, and the nano-preparation obtained in the example had an average particle size of 283 ± 13.8nm, a coating rate: ABT-263 is 74.2 +/-6.5%, and DOX is 70.7 +/-5.7%.
Example 7
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is implemented according to the following steps:
step 1, weighing raw materials: weighing 60% of medical gelatin, 2.5% of ABT-263, 35.5% of hyaluronic acid and 2% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding 336mg of medical gelatin weighed into 10ml of water, soaking for 1 hour at 25 ℃, heating to 51 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 14mg of ABT-263 and 160 mu l of polysorbate-80 into 20ml of ethanol, then dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 4ml/min and a stirring speed of 300rpm, 300 mu l of 25% glutaraldehyde solution is added after the solution is turbid, the stirring speed is increased to 800rpm, and the solution is reacted for 4 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion solution, removing the ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion solution through dialysis, filling the nano drug-loaded particle dispersion solution into a dialysis bag with the molecular weight cutoff of 3.5kDa, using pure water as an external solution, the dialysis time is 20 hours, replacing the external solution for 5 times, and finally using water to fix the volume to 20 ml;
step 4, slowly dropping 11.2ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 6 ml/min, the stirring speed is 1400rpm, stirring for 20min, adding 240 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 4 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 55mg of EDC and 5mg of NHS, stirring uniformly, standing for 6 hours, then filling the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 24 hours, the external liquid is replaced for 5 times to remove impurities and solvents, and then, under the stirring state, dropwise adding the ABT-263 drug-loaded nanoparticle dispersion liquid obtained in the step 3 at a speed of 4ml/min, at a stirring speed of 360rpm, and reacting at room temperature for 8 hours;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-80 ℃, the pre-freezing time is 24 hours, the drying is carried out by using a freeze dryer, and the pressure is controlled to be lower than 0.1 pascal.
The properties of the partially dried nano-formulation powder obtained in this example were analyzed by dissolving it in sterilized purified water and measuring its particle size using a malvern particle sizer, and the nano-formulation obtained in this example had an average particle size of 279 ± 15.8nm and a packing rate: ABT-263 is 73.5 +/-7.4%, and DOX is 71.8 +/-8.1%.
Example 8
A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is implemented according to the following steps:
step 1, weighing raw materials: weighing 52.5 percent of medical gelatin, 2.5 percent of ABT-263, 42 percent of hyaluronic acid and 3 percent of adriamycin according to the mass percent, wherein the sum of the mass percent of the components is 100 percent;
step 2, adding 315mg of medical gelatin into 10ml of water, soaking for 1 hour at 25 ℃, heating to 50 ℃, stirring for 20 minutes, and cooling to 30 ℃;
step 3, dissolving 10mg of ABT-263 and 120 mu l of polysorbate-80 into 15ml of ethanol, dropwise adding the solution into the stirred gelatin solution prepared in the step 2, wherein the ABT-263 ethanol solution is dropwise added at a speed of 3 ml/min and a stirring speed of 400rpm, 300 mu l of 25% glutaraldehyde solution is added after the solution is turbid, the stirring speed is increased to 9000rpm, and the solution is reacted for 4 hours in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion liquid, removing the ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion liquid through dialysis, filling the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with molecular weight cut-off of 3.5kDa, using pure water as an external liquid, carrying out dialysis for 18 hours, replacing the external liquid for 3 times, and finally using water to fix the volume to 19 ml;
step 4, slowly dropping 1.2ml of adriamycin aqueous solution with the concentration of 10mg/ml into 100ml of hyaluronic acid aqueous solution with the concentration of 2.5mg/ml, wherein the dropping speed of the adriamycin aqueous solution is 8 ml/min, the stirring speed is 1200rpm, stirring for 20min, then adding 300 mu l of glutaraldehyde solution with the concentration of 25%, and keeping out of the sun for 2 h to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the pH value of the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to 4 by using glacial acetic acid, then adding 45mg of EDC and 4.5mg of NHS, stirring uniformly, standing for 4 hours, then loading the nano drug-loaded nanoparticle dispersion liquid into a dialysis bag with the molecular weight cutoff of 100kDa, taking pure water as an external liquid, wherein the dialysis time is 12 hours, the external liquid is replaced for 3 times to remove impurities and solvents, then dropwise adding the ABT-263 drug-loaded gelatin nanoparticle dispersion liquid obtained in the step 3 under the stirring state, wherein the dropwise adding speed is 3 ml/min, the stirring speed is 200rpm, and reacting for 4 hours at room temperature;
and 6, filtering the nano drug-loaded particle dispersion liquid to remove non-soluble impurities, microorganisms and the like, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder, wherein the pre-freezing is carried out at the ultralow temperature of-80 ℃, the pre-freezing time is 18 hours, the drying is carried out by using a freeze dryer, and the pressure is controlled to be lower than 0.1 pascal.
Part of the nano-preparation dry powder obtained in the example was dissolved in sterilized pure water, and the particle size was measured by a malvern particle sizer to analyze the properties, and the nano-preparation obtained in the example had an average particle size of 302 ± 15.1nm and a coating rate: ABT-263 is 75.2 +/-7.1%, and DOX is 70.9 +/-3.5%.
The particle size distribution and stability of the doxorubicin, ABT-263 and doxorubicin dual-drug nanocarrier prepared in example 1 were measured, and morphological characteristics thereof were observed by a transmission electron microscope. The results obtained show that: the nanometer preparation has the advantages of uniform morphological characteristics, good monodispersity, high stability, and strong drug encapsulation ability, and is shown in fig. 2, fig. 3, and fig. 4. The in vitro cytotoxicity effect of the adriamycin, ABT-263 and adriamycin double-drug nano-carrier is detected, and the result shows that the adriamycin, ABT-263 and adriamycin double-drug nano-carrier has obvious cytotoxicity for human breast cancer cells MCF-7, human gastric cancer cells SGC7901 and human prostate cancer cells PC-3, and the result is shown in figure 5. The intervention is carried out on a nude mouse SGC7901 tumor-bearing model, and the result shows that the nano preparation can effectively inhibit the tumor growth, and the reference is made in figure 6.
Preliminary experiments show that the two medicines can play a role in the synergistic treatment of human breast cancer cells MCF-7, human gastric cancer cells SGC7901 and human prostate cancer cells PC-3, but the in-vivo synergistic treatment cannot be effectively exerted due to the difference of physicochemical properties, the repeatability of results cannot be ensured by a conventional emulsification and solubilization method, and the quality of the obtained preparation is difficult to control. The gelatin used in the invention is a widely used medicinal auxiliary material, which can effectively increase the dispersity of ABT-263 in water, and the hyaluronic acid has a certain tumor targeting property except being used as a packing material. According to the invention, based on earlier-stage experience, key parameters such as reagent proportion, preparation process and the like are repeatedly tested and modified through a large number of experiments, the defects of insufficient stability and poor repeatability in the preparation process are overcome, and the optimal preparation process is finally obtained. Ensures the quality control of the preparation.

Claims (6)

1. A preparation method of an adriamycin and ABT-263 double-drug nano-carrier is characterized in that the adriamycin and ABT-263 double-drug nano-carrier is composed of the following effective components of raw materials by mass percent: 52-60% of medical gelatin, 1.5-3% of ABT-263, 35-42% of hyaluronic acid and 1.5-3% of adriamycin, wherein the sum of the mass percentages of the components is 100%, and the molecular weight of the hyaluronic acid is 6000-8000 Dalton, and the preparation method specifically comprises the following steps:
step 1, weighing 52-60% of medical gelatin, 78-3% of ABT-2631.5, 35-42% of hyaluronic acid and 1.5-3% of adriamycin according to the mass percentage, wherein the sum of the mass percentages of the components is 100%;
step 2, adding the weighed medical gelatin into 10ml of water, soaking for a period of time, heating and stirring, and cooling to 30 ℃;
step 3, dissolving ABT-263 and polysorbate-80 in ethanol, dropwise adding the solution into the gelatin solution prepared in the step 2, stirring until the solution is turbid, adding a glutaraldehyde solution, increasing the stirring speed, reacting for a period of time in a dark place to obtain ABT-263 gelatin drug-loaded nanoparticle dispersion, removing an ethanol solvent, glutaraldehyde and other soluble impurities in the dispersion through dialysis, and finally adding water to a constant volume of 18-22 ml;
step 4, slowly dropping the adriamycin aqueous solution into the hyaluronic acid aqueous solution, stirring for a period of time, adding a glutaraldehyde solution, and keeping out of the sun for a period of time to obtain adriamycin drug-loaded nanoparticles;
step 5, adjusting the adriamycin drug-loaded nanoparticle dispersion liquid obtained in the step 4 to pH 4 with glacial acetic acid, then adding EDC and NHS, stirring uniformly, standing for a period of time, dialyzing the nano drug-loaded nanoparticle dispersion liquid to remove impurities and a solvent, then dropwise adding the ABT-263 gelatin drug-loaded nanoparticle dispersion liquid obtained in the step 3 in a stirring state, stirring, and reacting at room temperature;
and 6, filtering the nano drug-loaded particle dispersion liquid finally obtained in the step 5 to remove non-soluble impurities and microorganisms, then subpackaging in equal amount, and then pre-freezing, drying and removing water to obtain the final nano preparation dry powder.
2. The method for preparing the adriamycin and ABT-263 dual-drug nano-carrier in the step 2 is characterized in that the gelatin used in traditional Chinese medicine is 300-500 mg, the soaking water temperature is controlled at 25 ℃, the soaking time is 1 hour, the heating is carried out to 50-55 ℃, and the stirring time is 20 min.
3. The method for preparing the adriamycin and ABT-263 double-drug nano-carrier of claim 1, wherein in the step 3, 10-15 mg of ABT-263, 100-200 μ l of polysorbate-80, 12-20ml of ethanol, 25% of glutaraldehyde solution, 200-400 μ l in volume, 2-4 ml/min of ABT-263 ethanol solution dropping speed, 200-400 rpm of stirring speed, 800-1000 rpm of stirring speed, 2-4 hours of photophobic reaction duration, and the nano drug-loaded particle dispersion liquid is filled into a dialysis bag with molecular weight cutoff of 3.5kDa by dialysis, pure water is used as external liquid, and the dialysis time is 12-24 hours, during which the external liquid is replaced for 3-5 times.
4. The method for preparing the adriamycin and ABT-263 double-drug nano-carrier of claim 1, wherein in the step 4, the concentration of the adriamycin aqueous solution is 10mg/ml, the addition amount is 1-1.5 ml, the concentration of the hyaluronic acid aqueous solution is 2-3 mg/ml, the volume is 100ml, the dropping speed of the adriamycin aqueous solution is 5-10 ml/min, the stirring speed is 800-1500 rpm, the stirring time is 20min, the concentration of the glutaraldehyde solution is 25%, the addition amount is 200-400 μ l, and the duration of keeping out of the sun is 2-4 hours.
5. The method for preparing the adriamycin and ABT-263 double-drug nano-carrier of claim 1, wherein the amount of EDC in the step 5 is 40-60 mg, the amount of NHS is 4-6 mg, the standing time is 4-6 hours, the dialysis is to pack the nano drug-loaded particle dispersion liquid into a dialysis bag with the cut-off molecular weight of 100kDa, pure water is used as an external liquid, the dialysis time is 12-24 hours, the external liquid is replaced 3-5 times in the dialysis process, the acceleration of ABT-263 gelatin drug-loaded nanoparticle dispersion liquid drop is 2-4 ml/min, the stirring speed is 200-400 rpm, and the room-temperature reaction time is 4-8 hours.
6. The method for preparing the adriamycin and ABT-263 dual-drug nano-carrier of claim 1, wherein the pre-freezing in the step 6 is performed at an ultra-low temperature of-70 ℃ to-80 ℃, the pre-freezing time is 12-24 hours, and the drying is performed by using a freeze dryer under a pressure of less than 0.1 pascal.
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